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E. De Baere, B. D'haene, C. Attanasio, D. Beysen, E. Lemire, B. Lorenz, P. Bouchard, A. De Paepe, J. Dekker, S. Antonarakis; A Distant Regulatory 7.4 Kb Deletion Upstream of FOXL2 Causes BPES. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4119.
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An increasing number of studies focus on the potential function of non protein-coding elements of the human genome. Some of them are conserved non-coding sequences (CNCs) that can act as cis-elements and can regulate the spatiotemporal expression of developmental genes. FOXL2 encodes a forkhead transcription factor involved in the development of eye(lid) and ovary. Several types of mutations lead to the blepharophimosis syndrome (BPES): intragenic mutations (70%), gene deletions (10%) and long-range 3’ and 5’ microdeletions (4%), delineating a smallest region of deletion overlap (SRO) of 126 kb. The aim of the study was to explore the regulatory domain of FOXL2 in 57 BPES patients not carrying intragenic FOXL2 mutations nor gene deletions.
Microarray based comparative genome hybridization (array CGH), quantitative PCR (qPCR) and targeted sequencing of CNCs mapping within the SRO was performed in these patients. Deletion junction PCR and sequencing. Chromosome conformation capture (3C) of FOXL2 region. Luciferase assays of CNCs in cellular systems.
Apart from 3 novel "conventional" microdeletions, a de novo 7.4 kb deletion was found at 283 kb 5’ to FOXL2 in a typical BPES patient, thus delineating a much smaller SRO. Sequence analysis of the breakpoints allowed to reveal the underlying mechanism. Interestingly, this deletion contains a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs with presumed regulatory potential. Chromosome conformation capture (3C) revealed that three putative cis-regulatory fragments upstream of FOXL2 one of which harbours the reduced SRO, come in close vicinity to the FOXL2 core promoter.
We demonstrated that a distant 7.4 kb deletion leads to BPES, being the smallest fully characterized regulatory deletion ever described in human monogenic disease. Our study redefines a tissue-specific cis-regulatory domain of FOXL2 and emphasizes the importance of its integrity in human. Finally, CNCs were shown to be targets contributing to the molecular pathogenesis of human disease, which has significant implications towards mutation screening in human disease and eye development disorders in particular.
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